Dna construct for assessing thymic function activity and therapeutical uses thereof

ABSTRACT

The present invention relates to a DNA construct for in vivo expression in an excision DNA circle created by DNA recombination machinery in T cells from a non-human mammal which comprises two recombination signal sequences (RSS) consensus sequences flanking a promoter, an enhancer and a reporter gene, wherein the excision DNA circle is diluted out after cellular division and the excision DNA circle is detected by expression of the reporter gene and the detection is indicative of thymic function activity of the mammal. The present invention also relates to a T cell transiently transfected with the DNA construct of the present invention, the cell expressing quantifiable levels of reporter gene for green fluorescent protein (GFP) for determining enhancing/decreasing thymic exportation, a mammal comprising the DNA construct of the present invention and methods thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a DNA construct for assessing thymic functionactivity of a mammal, a DNA construct for screening drugs enhancingand/or decreasing thymic function of a mammal, and a method fordetecting and/or isolating T cells recently emigrated from the thymusamong others.

2. Description of Prior Art

The human thymus is responsible for the differentiation of immaturethymocytes into mature T lymphocyte expressing either the CD4 or the CD8molecule. During this process, thymocytes rearrange their genomic DNA atthe T cell receptor (TCR) α and β loci to generate TCR molecules thatwill be further selected during positive/negative selection. TCR generearrangement, mediated by recombination activating genes (RAG) 1 and 2,leads to the generation of stable TCRα and β recombination circles(TRECs) that do not replicate and that are diluted out during subsequentcellular proliferation. Each type of gene rearrangement event (δRec→ψJα,Vα→Jα, Dβ→Jβ and Vβ→DβJβ) generates a unique TREC that will have adistinct primary nucleotide sequence. Using PCR-based assays, severalgroups have shown that it is possible to evaluate the frequency of TRECsin T cell populations. These extrachromosomal circular DNA moleculeswere shown to be at a very high frequency in FACS-purifiedCD4⁺CD45RA⁺CD62L⁺ (naïve) T cells (Poulin, J.-F. et al., J. Exp.Med.,1999) and are now considered surrogate markers of recent thymicemigrants (RTEs) (Douek, D. C. et al., Nature, 1998). TRECquantification has become a direct indicator of ongoing thymopoiesis(Haynes, B. F. et al., Ann. Rev. Immunol., 2000).

At this date, no exclusive cell surface molecule specific to the human,macaque or mouse RTE population has been identified. In the chicken,chT1, an Ig-like molecule, was shown to be highly expressed on chickenthymocytes and quickly down-regulated upon thymocyte exportation(peripheral expression half-life of 2-3 weeks). Current assessment ofhuman and macaque thymic function is performed by evaluating TRECfrequencies in total genomic DNA or total cell lysate from T cellpopulations, which both lead to cell death. Thus, studies aiming atdiscovering exclusive functional and phenotypic properties of RTEs areimpossible until they can be accurately FACs-purified.

It would be highly desirable to be provided with the generation of a DNAconstruct model in which only TREC-containing T lymphocytes (e.g. RTEs)would express high levels of the green fluorescent protein (GFP), makingthem easily identifiable using conventional FACS technology. Thereby,mouse thymic function would be quantifiable by FACS analysis making themouse of the present invention a perfect model for the screening ofmolecules potentially playing a role in enhancing/decreasing thymicexportation.

SUMMARY OF THE INVENTION

In accordance with the present invention there is provided a DNAconstruct for in vivo expression in an excision DNA circle created byDNA recombination machinery in T cells from a non-human mammal whichcomprises two recombination signal sequences (RSS) consensus sequencesflanking a promoter, an enhancer and a reporter gene, wherein saidexcision DNA circle is diluted out after cellular division and theexcision DNA circle is detected by expression of the reporter gene andthe detection is indicative of thymic function activity of the mammal.

The DNA construct in accordance with a preferred embodiment of thepresent invention for screening drugs enhancing and/or decreasing thymicfunction, wherein an increase of detection level being indicative of adrug enhancing thymic function and wherein a decrease of detection levelbeing indicative of a drug decreasing thymic function, wherein theincrease or decrease is compared to thymic function of the mammal priorto administration of drug.

The DNA construct in accordance with a preferred embodiment of thepresent invention, wherein the RSS consensus sequences are sequencesrecognized by proteins recombination activating genes (RAG)1 and RAG2.

The DNA construct of the present invention as set forth in FIG. 1.

In accordance with the present invention, there is provided a T celltransiently transfected with the DNA construct of the present invention,the cell expressing quantifiable levels of reporter gene for greenfluorescent protein (GFP) for determining enhancing/decreasing thymicexportation.

The cell in accordance with a preferred embodiment of the presentinvention, wherein the DNA construct is introduced to the cell using avector selected from the group consisting of: retroviral vector,recombinant vaccinia vector, recombinant pox virus vector, poliovirus,influenza virus, adenovirus, adeno-associated virus, herpes and HIV.

The cell in accordance with a preferred embodiment of the presentinvention, wherein the DNA construct is introduced to the cell using aphysical method selected from the group consisting of: lipofection,direct DNA injection, microprojectile bombardment, electroporation,liposomes and DNA ligand.

In accordance with the present invention, there is provided a non-humanmammal for in vivo screening molecules enhancing and/or decreasingthymic function in a subject, comprising a cell subtype from a non-humantransfected with the DNA construct of the present invention, wherein thecell subtype after differentiation express quantifiable levels ofreporter gene for determining enhancing/decreasing thymic exportationcompared to thymic function prior administration of the molecules.

The mammal in accordance with a preferred embodiment of the presentinvention, wherein the cell is precursor of T lymphocyte.

The mammal in accordance with a preferred embodiment of the presentinvention, wherein the molecule is a potential modulator of thymicactivity.

The mammal in accordance with a preferred embodiment of the presentinvention, wherein the mammal is selected from the group consisting ofmouse, rat, chimpanzee and macaque.

In accordance with the present invention, there is provided a method fordetecting recent thymic emigrant (RTE), the method comprising the stepsof:

-   -   generating a transgenic mammal harboring the DNA construct of        the present invention into its genome;    -   isolating lymphocytes from organ samples taken from the mammal;    -   analyzing the lymphocytes for detecting presence of cells        expressing the reporter gene indicative of RTE.

In accordance with the present invention, there is provided a method forisolating RTE, the method comprising the steps of:

-   -   generating a transgenic mammal harboring the DNA construct of        the present invention into its genome;    -   isolating lymphocytes from organ samples taken from the mammal;    -   analyzing the lymphocytes for detecting presence, of cells        expressing the reporter gene indicative of RTE;    -   isolating the reporter gene expressing cells to obtain RTE.

The method in accordance with a preferred embodiment of the presentinvention, wherein the analyzing is performed by FACS analysis.

In accordance with the present invention, there is provided a method forin vivo quantification of thymopoiesis in a mammal, the methodcomprising the steps of:

-   -   generating a transgenic mammal harboring the DNA construct of        the present invention into its genome;    -   isolating lymphocytes from organ samples taken from the mammal;    -   quantifying the amount of cells expressing the reporter gene        from the lymphocytes    -   wherein the amount of cells expressing the reporter gene is        indicative of thymopoiesis in a mammal.

The method in accordance with a preferred embodiment of the presentapplication, wherein the quantifying is performed by FACSquantification.

In accordance with the present invention, there is provided a method foridentifying a RTE phenotype, the method comprising the steps of:

-   -   generating a transgenic mammal harboring the DNA construct of        the present invention into its genome;    -   isolating lymphocytes from organ samples taken from the mammal;    -   correlating expression of cytoplasmic and/or membrane bound        molecule to a RTE phenotype.

The method in accordance with a preferred embodiment of the presentinvention, wherein the correlation is performed by FACS analysis and/orimmunostrip assay.

In accordance with the present invention, there is provided a method formonitoring homeostasis of the RTE compartment in the mammal of thepresent invention, the method comprising the steps of:

-   -   generating a transgenic mammal harboring the DNA construct of        the present invention into its genome;    -   correlating expression of cells of the mammal having his thymus        ablated to a homeostasis of the RTE compartment in the mammal.

In accordance with the present invention, there is provided a method formonitoring homeostasis of the RTE compartment in the mammal of thepresent invention, the method comprising the steps of:

-   -   generating a transgenic mammal harboring the DNA construct of        the present invention into its genome;    -   administering anti-human CD4 monoclonal antibodies to the        mammal;    -   correlating expression of cells to a homeostasis of the RTE        compartment in the mammal.

In accordance with the present invention, there is provided a method formonitoring homeostasis of the RTE compartment in the mammal of thepresent invention, the method comprising the steps of:

-   -   generating a transgenic mammal harboring the DNA construct of        the present invention into its genome;    -   transferring reporter gene expressing cells of said mammal into        syngenic recipient, said recipient having been thymectomized,        irradiated or tolerized for said reporter gene.

In accordance with the present invention, there is provided a method fordetection of extrathymic T cell production in a mammal, the methodcomprising the steps of:

-   -   generating a transgenic mammal harboring the DNA construct of        the present invention into its genome;    -   eliminating thymic cells expressing the reporter gene in the        mammal; and    -   correlating neo-synthesized reporter gene expressing cells with        extrathymic T cell production in the mammal.

The method in accordance with a preferred embodiment of the presentinvention, wherein elimination of thymic cells expressing the reportergene comprises thymectomy and administration of anti-human CD4antibodies.

The method in accordance with a preferred embodiment of the presentinvention, wherein correlating neo-synthesized GFP+ cells compriseslongitudinal FACS analysis.

For the purpose of the present invention the following terms are definedbelow.

The term “reporter gene” is intended to mean a GFP gene or anydetectable gene that could be substituted, it may also have the sameactivity, it may also intend any fluorescent, radioactive label and anynon fluorescent membrane-bound protein detected by a specific monoclonalor polyclonal antibody coupled to any label.

In the present invention, any strong promoter from viruses or eukaryoticcells could replace the promoter. Also, the enhancer could be replacedwith any other strong enhancer. It is well known in the art what astrong promoter and a strong enhancer are and one skilled in the artwill easily know what promoter and enhancer may be used to realize thepresent invention.

Also, even if the recombination signal sequences (RSS) disclosed in thepresent application are the preferred embodiment realized by theApplicant, RSS can still be “point-mutated” and replaced with some lessefficient one and be functional. One skilled in the art would know howto proceed with such a mutation without affecting functionality of theRSS.

It is also understood that in order to target the recombinationmachinery where the transgene of the present application inserteditself, elements were incorporated ensuring that during TCRαrearrangement recombination of the transgene will occur. Exchangingthose elements for TCRβ, TCRγ or TCRδ specific elements woulddefinitively help tracking down other type of “newly produced” T cells(γδ if TCRγ or TCRδ elements for example).

In the present application, it is also understood that the mouse RTEsexpressing GFP synthesize a truncate version of human CD4 molecule wherethe cytoplasmic domain of hCD4 is lacking, but that this gene could bereplaced with any gene issued from any living organism without affectingthe functionality of the present invention. This gene needs to have atransmembrane region and it could also be a soluble protein or peptidefused to a transmembrane region or a transmembrane protein.

All references herein are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the pre-rearrangement construct;

FIG. 2 illustrates the post-rearrangement construct;

FIG. 3 illustrates the efficiency of expression of thepost-rearrangement constructs;

FIG. 4 illustrates a western blot analysis demonstrating that M12 andDr3 cell lines express variable level of the Rag 2 protein, while the1.8 cell line does not express Rag 2 protein; and

FIG. 5 illustrates flow cytometry assays performed 48 hours followingthe transfection of cell lines by pre-rearrangement constructs, wherethe GFP protein was measured.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a DNAconstruct for expression in an excision circle created by T and/or Bcells DNA rearrangement, this circle being transitory and disappearafter cellular division. This DNA construct is useful for screeningdrugs enhancing and/or decreasing thymic function.

Given the fact that TREC are considered surrogate markers of thymicfunction irrespective of cell surface molecule expression, a DNAconstruct was engineered, this construct being used for introduction ina cell or a mammal as a dsDNA transgene. This transgene is bearingoptimized recombination signal sequences (RSS) and TCRα locus-specificrecombination elements that recruits the RAG machinery expressed duringthymocyte ontogeny (FIG. 1). This rearrangement event generates a uniqueexcision circle in which the viral SRα promoter is in-frame of the GFPgene, thereby making TREC-containing cells (e.g. newly produced T cells)GFP+.

A wide variety of methods may be utilized in order to deliver the DNAconstruct of the present invention to a warm-blooded animal orbiological preparation. For example, within one embodiment of theinvention, the vector construct is inserted into a retroviral vector,which may then be administered directly into a warm-blooded animal orbiological preparation. Representative examples of suitable retroviralvectors and methods are described in more detail in the following U.S.patents and patent applications, all of which are incorporated byreference herein in their entirety: “DNA constructs for retroviruspackaging cell lines”, U.S. Pat. No. 4,871,719; “RecombinantRetroviruses with Amphotropic and Ectotropic Host Ranges”, PCTPublication No. WO 90/02806; and “Retroviral Packaging Cell Lines andProcesses of Using Same”, PCT Publication No. WO 89/07150.

DNA construct may also be carried by a wide variety of other viralvectors, including for example, recombinant vaccinia vectors (U.S. Pat.Nos. 4,603,112 and 4,769,330), recombinant pox virus vectors (PCTPublication No. WO 89/01973), poliovirus (Evans et al. Nature,339:385-388 (1989); and Sabin, J. Biol. Standardization, 1:115-118(1973)); influenza virus (Luytjes et al., Cell, 59:1107-1113 (1989);McMichael et al., N. Eng. J. Med., 309:13-17 (1983); and Yap et al.,Nature, 273:238-239 (1978)); adenovirus (Berkner, Biotechniques,6:616-627 (1988); Rosenfeld et al., Science, 252:431-34 (1991));adeno-associated virus (Samulski et al., J. Virol., 63:3822-3828 (1989);Mendelson et al., Virol., 166:154-165 (1988)); herpes (Kit, Avd. Exp.Med. Biol., 215:219-236 (1989)); and HIV (Poznansky, J. Virol.,65:532-536 (1991)).

In addition, DNA construct may be administered to warm-blooded animalsor biological preparations utilizing a variety of methods, including,without limitation, lipofection (Felgner et al. Proc. Natl. Acad. Sci.USA, 84:7413-7417 (1989), direct DNA injection (Acsadi et al., Nature,352:815-818 (1991)); microprojectile bombardment (Williams et al., PNAS,88-2726-2730 (1991)); liposomes (Wang et al., PNAS, 84:7851-7855(1987)); CaPO4 (Dubensky et al., PNAS, 81:7529-7533 (1984)); or DNAligand (Wu et al., J. Biol. Chem., 264:16985-16987 (1989)).

Using the warm-blooded animal or biological preparation containing theDNA construct, it is possible to isolate real naïve cell irrespective ofcell surface molecule expression patterns. A fraction of RTE is detected(those that have not yet loss their TREC due to the dilution effect).

It is well documented that B cells also express the same recombinationmachinery at some point in their development. This could lead to in vivotransgene rearrangement in B cells, making them fluorescent and generate“false positive”. In order to circumvent that, TCRα locus-specificelements are inserted downstream of the transgene (Winoto A, BaltimoreD. EMBO J. 1989 March;8(3):729-33. Winoto, A. and Baltimore, D. Cell 59(4), 649-655 (1989)), ensuring the cell type specificity of therearrangement. The function of these elements is to ensure that thetransgene of the present invention becomes accessible to the RAGmachinery during T cell ontogeny.

In order to ensure a tight regulation of GFP fluorescence emissiondespite the integration site in the host chromosome, stop codons areinserted 3′ of the GFP gene (between the GFP and TCRα locus-specificelements). Furthermore, the endogenous ATG start codon from the GFP geneis “knocked-out” and inserted within the 23 bp spacer located within oneRSS (FIG. 1). Given this, not leak in GFP fluorescence from cells thatdo not express the RAG machinery at one point in their life isanticipated.

Also, in order to study the in vivo replenishment rate of RTE, anIRES-hCD4 fragment is inserted in the transgene. Given the fact thatthis fragment is located on the excised DNA circle, downstream from theGFP but upstream from the polyadenylation site, this hCD4 protein willbe produced together with GFP. Thus, the injection of an antibodydirected against hCD4 would deplete (via the complement pathway) allGFP+ T cells (e.g. RTEs). This fragment may be viewed as a “resetbutton” for RTEs production.

These are the elements generated using the PCR technology and standardmolecular biology techniques.

-   -   Backbone cloning vector: Stratagene pBSKS (Bluescript)    -   GFP gene: Clontech peGFP-C1    -   TCRα enhancer 1 and silencer 1: mouse genomic DNA    -   Silencer 2: mouse genomic DNA    -   SRα0 viral promoter (Takebe et al, 1988. Mol Cell Biol 8466: 72)    -   Junk 1, 2 and 3: human genomic DNA    -   CD3 enhancer: mouse genomic DNA    -   IgM enhancer: mouse genomic DNA    -   CD3δ promoter: mouse genomic DNA    -   IRES-hCD4: cloned IRES and human truncated CD4 (no cytoplasmic        tail)        “Proof-of-Concept” Validation

dsDNA constructs that exactly simulate the end product of therearrangement events (see FIG. 2) were designed and generated. Theseconstructs are referred to as the “post-rearrangement” constructs. Thevarious elements constituting these constructs were sequentiallyintroduced in pBSKS (Bluescript) cloning vector, in which we hadpreviously exchanged the multiple cloning site (MCS) region with onebearing the required restriction sites. This allowed us to optimize thepromoter/enhancer combination that will be used in the final constructs(Table 1).

An example of GFP expression following transient transfection of 50 μgof the “post-rearrangement constructs” in 5×10⁶ Jurkat E6.1 cell line isshown in FIG. 3. As shown in Table 1, variable GFP expression wasobserved using the different constructs. Transfection experimentsperformed in Jurkat E6.1 cell line were able to demonstrate that the Srαpromoter, coupled to the CD3δ enhancer is the best combination toexpress GFP (underlined data) in the post-rearrangement constructs inthis particular T cell line (table 1a). As shown in table 1b, theefficiency of this combination was confirmed in DR3 cells thatconstitutively express the Rag1 and Rag2 proteins, and consequently wasused in the next series of experiments. TABLE 1 Optimization ofpromoter/enhancer combination a-Experiment 1 Jurkat-E6.1 % of GFP+ CellsMean Fluorescence pBSKS-MCS-JFR  0.07  7.48 peGFP-C1 64.04 1260.19 pSRα-CD3enh 35.44 914.69 pSRα-CD3enh 38.15 540.32 pSRα-CD3enh 31.84341.84 pSRα-μenh 26.19 276.92 pSRα-μenh 31.71 407.05 pCD3δ CD3enh 24.76214.18 pCD3δ CD3enh 24.54 163.17 pCD3δ-μenh 26.62 226.85 b-Experiment 2DR3 % of GFP+ Cells Mean Fluorescence no DNA  0.09  10.34 peGFP-C1 96.322599.87  pSRα-CD3enh 87.98 482.35 pSRα-CD3enh 81.85 408.39 pCD3δ-CD3enh32.50 183.53 pCD3δ-μenh 62.69 452.55 Experiment 3 DR3 % of GFP+ CellsMean Fluorescence pBSKS-MCS-JFR  0.08  10.75 peGFP-C1 97.35 3458.11 pCD3δ-CD3enh 39.42 389.67 pCD3δ-μenh 63.87 636.87 pSRα-CD3enh 97.941510.26 Legend to Table 1:Transfection experiments of the post rearrangement constructs.a) Four different DNA constructs were transfected into Jurkat-E6.1 cellline. GFP expression was observed for all construct, the pSRα-CD3enhcombination being the more efficient (underlined data).b) The same constructs were transfected into Rag1/2 expressing DR3 cellline. Again, the pSRα-CD3enh construct leads to higher GFP expressionlevels (underlined data). For these experiments, pBSKS-MCS-JFR is anegative control and peGFP-C1 is a positive control.

Once the optimal elements were identified (the SRα promoter and the CD3δenhancer), the non-rearranged dsDNA transgene (e.g. the“pre-rearrangement” construct) was synthesized and tested for itsability to recombine in vitro using RAG-1/2 expressing cell lines.

Several constructs were generated with the CD3δ or the SRα promoter Witheither the CD3δ or the μEnhancer. These DNA constructs were transfectedinto the 1-8, M12 and Dr3 cell lines that express variable level of theRag 2 protein, as demonstrated by western blot analysis (FIG. 4). Inthese cell lines, the expression of the GFP protein was measured by flowcytometry analysis 48 hours following transfection (FIG. 5). In 3independent experiments (Table 2), GFP was detected in all the Ragexpressing cell lines (i.e. M12 and DR3) while the 1.8 cell lineremained negative for the expression of GFP (Table 2a). Of note is thefact that the 1.8 cell line is able to express GFP when transfected withthe post-rearrangement constructs (Table 2a), demonstrating that thelack of expression is a consequence of the inability of these cells torearrange the pre-rearrangement construct due to the absence of Ragexpression.

These results were confirmed in several independant experiments (Table2b). Experiment 2 demonstrates that the expression of the GFP followingtransfection in the Rag-expressing cell line is due to rearrangement ofthe DNA construct and not to non-specific GFP transcription through anunknown promoter located 3′ of the GFP gene. This is demonstrated by GFPexpression in transfection experiments with Not I and Bgl II digestedDNA constructs. These restriction enzymes are able to digest thepre-rearrangement construct 5′ of the 5′-RSS and 3′ of the 3′-RSS, thusremoving parts of the construct susceptible to contain non-specificrecombination sequences or cryptic promoters.— TABLE 2 In vitroTransfection experiments a-Experiment # 1 Mean Fluo. of Cells Plasmids %of GFP+ Cells GFP+ cells 1-8 pBSKS-MCS-JFR 0.03 100.88 M12 pBSKS-MCS-JFR0.04 102.96 DR3 pBSKS-MCS-JFR 0.22 695.07 1-8 Post-pSRα-CD3enh 2.59 27.17 M12 Post-pSRα-CD3enh 47.81  211.43 DR3 Post-pSRα-CD3enh 89.25 756.52 1-8 Pre-pSRα-CD3enh 0.01  17.29 M12 Pre-pSRα-CD3enh 1.13  49.31DR3 Pre-pSRα-CD3enh 6.74 153.79 b-Experiment # 2 Mean Fluo. of CellsPlasmids % of GFP+ Cells GFP+ cells M12 pBSKS-MCS-JFR 0.46 NA DR3pBSKS-MCS-JFR 0.07 NA M12 Pre-pSRα-CD3enh 1.41  42.04 DR3Pre-pSRα-CD3enh 8.32 123.02 M12 Pre-SRα-CD3enh 2.56  42.22 (Not 1-BglII) DR3 Pre-SRα-CD3enh 15.01   79.54 (Not 1-Bgl II)Legend to table 2:Transfection experiments of the Pre-rearrangement construct.a) The Pre rearrangement construct (pSRα-CD3enh) was transfected intoseveral cell lines, expressing variable levels of Rag1/2 proteins.# GFP expression correlates to Rag expression. The positive control(pSRα-CD3enh - post rearrangement construct) is expressed in all testedcell lines.b) The expression of GFP is not due to non-specific transcription thoughan unknown promoter located 3′ of the GFP gene in the pre-rearrangementconstruct.# DNA construct was digested in order to excise the sequence susceptibleto be rearranged, following transfection, these digested constructs areable to express GFP showing that this expression is a consequence of DNArearrangement. # In these experiments the PBSKS-MCS-JFR plasmid was usedas a negative control while the post rearrangement constructPost-pSrαCD3enh served as a positive control.

EXAMPLE I Extensive Phenotypic Characterization of Mouse Recent ThymicEmigrants

In order to proceed with the identification of a mouse RTE-specificphenotype, GFP^(high) PBMC isolated from the mice is phenotypicallycharacterized using a multiple mouse monoclonal antibodies directedagainst CD4, CD3, CD8, TLA4, CD28, CD95, CD27, ICAM-1, α₄β₇ integrin,chemokine and hormone receptors (GM-CSF, c-kit).

EXAMPLE II Mice Crosses with Mouse Cytokine/Chemokine Knock-Out

As a novel way to determine the role of any cytokine (in this case IL-7)on thymopoiesis regulation, mice are crossed with the IL-7 knock-outmice given the fact that IL-7 plays an important role in themaintenance/survival of the naïve T cell compartment. The end-product ofthis crossing is a cytokine or chemokine knock-out mice in which RTE canbe detected, quantified and isolated.

EXAMPLE III Extrathymic Generation of T Cells

Hematopoietic stem cells (T cells precursors c-Kit⁺, Ly-6A/E⁺, Lin⁻)isolated from day 14 fetal liver of a mouse is infused in thymectomizedor sham-thymectomized irradiated syngenic and congenic mice.Longitudinal studies measuring the rate of appearance of GFP⁺ T cells isdone on both groups. If present, the identification of the organresponsible for de novo extrathymic production of T cells(gut-associated lymphoid tissue (GALT), spleen or possibly lymph nodes)will be identifiable by fluorescence detection.

EXAMPLE IV Determination of RTE Cell Activation Requirements

Recent thymic emigrants may need to undergo maturation steps beforebecoming real functional naïve T cells able to respond to antigens. Thisis fully compatible with recent experiments demonstrating that naïve Tcells can “homeostatically” proliferate without loosing their naïvephenotype. It is possible that these rounds of replication remodel thechromatin, making some transcriptionaly-inactive genes expressed (KieperW C, Jameson S C. Proc Natl Acad Sci USA. 1999 Nov. 9;96(23):13306-11.Goldrath A W, Bogatzki L Y, Bevan M J. J Exp Med. 2000 Aug.21;192(4):557-64.) To answer that, FACS-purified GFP^(High) T cells(e.g. “real” recent thymic emigrants) are stained with CFSE, a celldivision marker. RTE stimulation is done using anti-CD3 and anti-CD28antibodies and cytokines production monitored by FACS analysis. Withthis, the number of rounds of replication required for RTE to reachfunctional maturity can be determined.

EXAMPLE V Compartmentalization of RTE

Where do new cells go when they are produced? It is though that naïve Tcells go into lymph nodes (where potential antigens are likely to bepresented) once they are generated. In our model, tracking-down RTE canbe done using histological slides of various peripheral organs (lymphnodes, spleen, gut-associated lymphoid tissue). Infusion into normalmice of hematoipoietic stem cells previously isolated from the mousefollowed by histological studies help understanding the faith of de novoproduced T cells.

EXAMPLE VI Do RTE Contribute to the Maintenance/Re-Seeding of the SIVReservoir?

Ex vivo transfection of Macaca Mulata CD34+ precursors cells with ourtransgene followed by re-infusion in irradiated SIV-infected hostsbearing or not a thymus help investigating if RTE harbor SIV proviralDNA, thereby assessing the contribution of ongoing thymopoiesis to theSIV reservoir. Thymocytes were shown to be infected both in vivo and invitro. Again, GFP^(High) T cells are FACS-purified and SIV proviral DNAquantified using the LightCycler™ real-time on-line quantitative PCRtechnology available now in the lab. Of course, the mouse specificelements have to be replaced by their homologue in the macaque model.

Also, analogous experiments previously performed in mice can be done inthe macaque model using the same “pre-rearrangement” transgene.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. A DNA construct for in vivo expression in an excision DNA circle created by DNA recombination machinery in T cells from a non-human mammal which comprises two recombination signal sequences (RSS) consensus sequences flanking a promoter, an enhancer and a reporter gene, wherein said excision DNA circle is diluted out after cellular division and said excision DNA circle is detected by expression of said reporter gene and said detection is indicative of thymic function activity of said mammal.
 2. The DNA construct of claim 1 for screening drugs enhancing and/or decreasing thymic function, wherein an increase of detection level being indicative of a drug enhancing thymic function and wherein a decrease of detection level being indicative of a drug decreasing thymic function, wherein said increase or decrease is compared to thymic function of said mammal prior to administration of drug.
 3. The DNA construct of claim 1, wherein said RSS consensus sequences are sequences recognized by proteins recombination activating genes (RAG)1 and RAG2.
 4. A DNA construct of claim 1 as set forth in FIG.
 1. 5. A T cell transiently transfected with the DNA construct of claim 1, said cell expressing quantifiable levels of reporter gene for green fluorescent protein (GFP) for determining enhancing/decreasing thymic exportation.
 6. The cell of claim 5, wherein said DNA construct is introduced to said cell using a vector selected from the group consisting of: retroviral vector, recombinant vaccinia vector, recombinant pox virus vector, poliovirus, influenza virus, adenovirus, adeno-associated virus, herpes and HIV.
 7. The cell of claim 5, wherein said DNA construct is introduced to said cell using a physical method selected from the group consisting of: lipofection, direct DNA injection, microprojectile bombardment, electroporation, liposomes and DNA ligand.
 8. The cell of any one of claim 5, wherein said RSS consensus sequences are sequences recognized by proteins RAG1 and RAG2.
 9. The cell of any one of claim 5, wherein said DNA construct is a DNA construct as set forth in FIG.
 1. 10. A non-human mammal for in vivo screening molecules enhancing and/or decreasing thymic function in a subject, comprising a cell subtype from a non-human transfected with the DNA construct of claim 1, wherein said cell subtype after differentiation express quantifiable levels of reporter gene for determining enhancing/decreasing thymic exportation compared to thymic function prior administration of said molecules.
 11. The mammal of claim 10, wherein said cell is precursor of T lymphocyte.
 12. The mammal of claim 10, wherein said molecule is a potential modulator of thymic activity.
 13. The mammal of claim 10, wherein said mammal is selected from the group consisting of mouse, rat, chimpanzee and macaque.
 14. The mammal of claim 10, wherein said mammal is a mouse.
 15. The mammal of claim 10, wherein said RSS consensus sequences are sequences recognized by proteins RAG1 and RAG2.
 16. The mammal of claim 10, wherein said DNA construct is a DNA construct as set forth in FIG.
 1. 17. A method for detecting recent thymic emigrant (RTE), said method comprising the steps of:—generating a transgenic mammal harboring the DNA construct of claim 1 into its genome;—isolating lymphocytes from organ samples taken from said mammal;—analyzing said lymphocytes for detecting presence of cells expressing said reporter gene indicative of RTE.
 18. A method for isolating RTE, said method comprising the steps of:—generating a transgenic mammal harboring the DNA construct of claim 1 into its genome;—isolating lymphocytes from organ samples taken from said mammal;—analyzing said lymphocytes for detecting presence of cells expressing said reporter gene indicative of RTE;—isolating said reporter gene expressing cells to obtain RTE.
 19. The method of claim 17, wherein said analyzing is performed by FACS analysis.
 20. The method of claim 17, wherein said mammal is selected from the group consisting of mouse, rat, chimpanzee and macaque.
 21. The method of claim 17, wherein said mammal is a mouse.
 22. The method of claim 17, wherein said RSS consensus sequences are sequences recognized by proteins RAG1 and RAG2.
 23. The method of claim 17, wherein said DNA construct is a DNA construct as set forth in FIG.
 1. 24. A method for in vivo quantification of thymopoiesis in a mammal, said method comprising the steps of:—generating a transgenic mammal harboring the DNA construct of claim 1 into its genome;—isolating lymphocytes from organ samples taken from said mammal;—quantifying the amount of cells expressing said reporter gene from said lymphocytes wherein said amount of cells expressing said reporter gene is indicative of thymopoiesis in a mammal.
 25. The method of claim 24, wherein said quantifying is performed by FACS quantification.
 26. The method of claim 24, wherein said mammal is selected from the group consisting of mouse, rat, chimpanzee and macaque.
 27. The method of claim 24, wherein said mammal is a mouse.
 28. A method for identifying a RTE phenotype, said method comprising the steps of:—a transgenic mammal harboring the DNA construct of claim 1 into its genome;—isolating lymphocytes from organ samples taken from said mammal);—correlating expression of cytoplasmic and/or membrane bound molecule to a RTE phenotype.
 29. The method of claim 28, wherein said correlating is performed by FACS analysis and/or immunostrip assay.
 30. The method of claim 28, wherein said phenotype is the phenotype of a mammal selected from the group consisting of mouse, rat, chimpanzee and macaque.
 31. The method of claim 28, wherein said mammal is a mouse.
 32. A method for monitoring homeostasis of the RTE compartment in the mammal of claim 10, said method comprising the steps of:—generating a transgenic mammal harboring the DNA construct of claim 1 into its genome;—correlating expression of cells of said mammal having his thymus ablated to a homeostasis of the RTE compartment in said mammal.
 33. A method for monitoring homeostasis of the RTE compartment in the mammal of claim 10, said method comprising the steps of:—generating a transgenic mammal harboring the DNA construct of claim 1 into its genome;—administering anti-human CD4 monoclonal antibodies to said mammal; correlating expression of cells to a homeostasis of the RTE compartment in said mammal.
 34. A method for monitoring homeostasis of the RTE compartment in the mammal of claim 10, said method comprising the steps of:—generating a transgenic mammal harboring the DNA construct of claim 1 into its genome;—transferring reporter gene expressing cells of said mammal into syngenic recipient, said recipient having been thymectomized, irradiated or tolerized for said reporter gene.
 35. The method of claim 32, wherein said subject is selected from the group consisting of mouse and macaque.
 36. The method of claim 32, wherein said subject is a mouse.
 37. The method of claim 32, wherein said RSS consensus sequences are sequences recognized by proteins RAG1 and RAG2.
 38. The method of claim 32, wherein said DNA construct is a DNA construct as set forth in FIG.
 1. 39. A method for detection of extrathymic T cell production in a mammal, said method comprising the steps of:—generating a transgenic mammal harboring the DNA construct of claim 1 into its genome;—eliminating thymic cells expressing said reporter gene in said mammal; and—correlating neo-synthesized reporter gene expressing cells with extrathymic T cell production in said mammal.
 40. The method of claim 39, wherein elimination of thymic cells expressing said reporter gene comprises thymectomy and administration of anti-human CD4 antibodies.
 41. The method of claim 39, wherein correlating neo-synthesized GFP+ cells comprises longitudinal FACS analysis.
 42. The method of claim 39, wherein said mammal is selected from the group consisting of mouse and macaque.
 43. The method of claim 39, wherein said mammal is a mouse.
 44. The method of claim 18, wherein said analyzing is performed by FACS analysis.
 45. The method of claim 18, wherein said mammal is selected from the group consisting of mouse, rat, chimpanzee and macaque.
 46. The method of claim 18, wherein said mammal is a mouse.
 47. The method as claimed in claim 18, wherein said RSS consensus sequences are sequences recognized by proteins RAG1 and RAG2.
 48. The method as claimed in claim 18, wherein said DNA construct is a DNA construct as set forth in FIG.
 1. 49. The method of claim 33, wherein said subject is selected from the group consisting of mouse and macaque.
 50. The method of claim 34, wherein said subject is selected from the group consisting of mouse and macaque.
 51. The method of claim 33, wherein said subject is a mouse.
 52. The method of claim 34, wherein said subject is a mouse.
 53. The method of claim 33, wherein said RSS consensus sequences are sequences recognized by proteins RAG1 and RAG2.
 54. The method of claim 34, wherein said RSS consensus sequences are sequences recognized by proteins RAG1 and RAG2.
 55. The method of claim 33, wherein said DNA construct is a DNA construct as set forth in FIG.
 1. 56. The method of claim 34, wherein said DNA construct is a DNA construct as set forth in FIG.
 1. 